Gene Technology

Contents

• 1. Introduction• 2. Applications• 3. Insulin Production• 4.Use of fluorescent markers over antibiotic

markers.• 5. Importance of Promoters• 6.Social and Ethical Impacts of Gene

Technology• 7. Cystic Fibrosis

What is gene technology?•Genetic technology is the

manipulation of organisms' genes.

•The DNA of two different organisms can be combined together to create a new gene and hence change the protein that the cell synthesizes.

Applications of Gene Technology •In the mass-production of human insulin

•Medical diagnosis and treatment, e.g Cystic Fibrosis

•Production of GM food, e.g the Golden rice

•Improving crop resistance to insects and certain herbicides 

Insulin Production•One form of diabetes mellitus is caused by

the inability of the pancreas to produce insulin.

•Gene technology is used to produce human insulin.

•The gene coding for human insulin is inserted into a bacterium to make insulin.

Insulin Production•Steps in Insulin Production•1. Isolating the insulin gene•2. Preparation of vector•3. Inserting gene into vector•4. Inserting gene into bacteria•5. Identification of transformed bacteria•6. Cloning and Mass- production

1. Isolating the Insulin Gene• mRNA coding for synthesis of insulin is

extracted from β cells in human pancreas.

• Extracted mRNA is incubated with a mixture of free DNA nucleotides and the enzyme reverse transcriptase.

• Single strand of cDNA is then produced.

• Using DNA polymerase, the cDNA is converted to double stranded DNA.

Isolating Insulin gene• Non-coding DNA was added to the cDNA so that when

restriction enzymes could produce sticky ends.

• Restriction enzymes cut

• DNA at specific base sequences- their restriction sites.

Figure showing Restriction enzyme EcoR1

Preparation of Vector• Bacterial plasmid is used as vector.

• Plasmids often contain antibiotic resistance gene- serve as markers.

• The selected plasmid is cut using the same restriction enzyme used on the cDNA to have complementary sticky ends

Inserting gene into vector• The cut plasmid and the cut human DNA are

mixed together.

• Complementary bases in the sticky ends pair up.

• DNA ligase enzyme is added to link the nucleotide backbones and ensures that the gene is permanently added to the plasmid

• Recombinant DNA is then formed

Inserting gene into plasmid

• Figure showing complementary sticky ends pairing up to form Recombinant DNA

Inserting gene into bacteria• The plasmids containing human genes are mixed with

bacterial cells, E.coli.

• A small proportion of bacteria successfully take up the plasmid containing insulin gene.

• These are now termed as transformed bacteria.

Figure showing formation of recombinant DNA

Identification of transformed bacteria•The genetically modified bacteria need to

be separated from other bacterial cells.

•Bacteria are treated with antibiotic, e.g ampicilin.

•Only those that contain resistance gene can survive.

Identification of transformed bacteria

Cloning and Mass-production•The identified transformed bacteria,

containing the insulin gene are now grown at industrial scale in large scale to produce insulin.

Summary of human insulin production

Use of fluorescent markers over antibiotic markers

• The antibiotic resistance genes can potentially spread to other bacteria, that may produce pathogenic bacteria.

• Controlling the spread of pathogenic bacteria using antibiotic would be unsuccessful.

• As an alternative, enzymes that produce fluorescent substances are used which are:

- harmless - are easily stained

• The gene for the enzyme is inserted into the plasmid.Thus, identification of transformed bacteria is easier.

Promoters• Promoter is a region of DNA in bacterial cells that

controls the expression of each gene.

• A suitable promoter needs to be transferred along with the human insulin gene.

• The insulin gene is inserted next to a β-galactosidase gene in the plasmid, so that they share a promoter.

• When transformed bacteria is grown in a medium containing lactose, both genes are expressed.

Social and ethical implications of Gene Technology

1. Social Impact

• Deals mainly with the potential and actual impact of Gene Tech on human society and individuals.

• enhance crop yields and permit crops to be better adapted

• enhance the nutritional content of crops so that people are better fed

• production of more effective and cheaper medicines and treatments through genetic manipulation of microorganisms and agricultural organisms to make medicines and genetic manipulation of human cells and individuals (gene therapy)

Social Impact of Gene Technology• increase costs of seed and prevent seed from being

retained for sowing next year (by inclusion of genes to kill any seed produced this way) reducing food production

• reduce crop biodiversity by out-competing natural crops.

• cause antibiotics to become less useful and cause allergic reactions or disease in other unexpected ways

• damage useful materials such as oil or plastic in unexpected ways

2. Ethical Impacts of Gene technology • Involves the application of moral frameworks concerning the principles

of conduct on what might be be right or wrong.

• It is good to conduct such research to develop technologies that might improve nutrition, the environment or health.

• It is good to use this technology to increase producetion of food, to enhance the environment or improve health.

• It is wrong to continue such research when the potential impact of the technology is unknown and many aspects of it remain to be understood.

• It is wrong to use the results of such research even when the organisms are kept in carefully regulated environments such as sterile fermenters as the risks of the organisms or the genes they contain escaping are too great and unknown

Cystic Fibrosis (CF)•It is a genetic disease in which abnormally

thick mucus is produced in the lungs and other parts of body.

•Inherited disease

•Significantly decreases life expectancy,however with improved treatments such people are able to live longer.

•The average life span is now 35 years old.

Causes of Cystic Fibrosis• By a recessive allele of the gene that codes for a transporter

protein called CFTR.

• This transmembrane protein allow chloride ions to pass out of cells, e.g in alveoli.

• Mutations in the CFTR gene have produced several different defective alleles which are recessive

• These defective alleles can be inherited to several generations.

• Someone with heterozygous allele for the CFTR gene is a carrier of the disease.

• Someone with recessive homozygous alleles for CFTR genes is affected by CF

• Figure showing inheritance of recessive faulty CFTR genes

Effects of Cystic Fibrosis• The faulty version of CFTR protein can no longer act

properly as chloride ion transporter.

• In a person with CF, there reduced Cl- transport through cell membranes.

This leads to production of abnormally thick, sticky mucus. Figure showing CFTR protein

Effects of Cystic Fibrosis• Can cause bacterial infections in lungs as the thick

mucus is difficult to be removed and bacteria breed in it.

• The mucus may block the pancreatic duct, preventing amylase and protease enzymes from reaching the small intestine.

• The mucus may block the sperm ducts, causing male infertility and may slow the progress of eggs and sperm through the oviducts, reducing female fertility.

•Social end ethical implications of G.T•Use of electrophoresis•Causes and Symptoms of CF•Treating CF with G.T